From Drosophila to humans, the Wnt signaling pathway plays a crucial role in cell fate determination and patterning during embryonic development as well as in adults and in disease processes. In addition to the ubiquitous cell polarity along the apical-basal axis, many epithelial tissues and organs also display polarization within the plane of epithelium referred to as Wnt/planar cell polarity (PCP). A core of Wnt/PCP signaling proteins, including the Frizzled (Fz) receptor and Dishevelled (Dsh;Dvl in mammals), participate in tissue development in the Drosophila wing. Subsequent work in vertebrates has determined that the Wnt/PCP pathway is evolutionarily conserved. Plasma membrane targeting of Dvl is a critical step in both canonical ([unreadable]-catenin) and Wnt/PCP signaling. In mammals, Dvl exists in three isoforms (Dvl1, Dvl2, and Dvl3) of putative redundant function with each consisting of three conserved domains known as DIX, PDZ, and DEP. Whereas the Dvl PDZ domain mediates binding to the Fz receptor, the DEP domain facilitates targeting of Dvl to the plasma membrane by a yet unclear mechanism. In vivo studies of the Wnt/PCP pathway established that the interaction of Dvl1 with the Fz receptor is both pH- and charge-dependent. Furthermore, a weak interaction of Dvl1 DEP domain with anionic phospholipids, including phosphatidic acid (PA), was demonstrated by biochemical assays and mutagenesis analyses. Using nuclear magnetic resonance (NMR) spectroscopy, the investigator has identified and mapped the Dvl2 DEP domain residues involved in PA recognition. Thus, Specific Aim 1 of the project is to characterize the mechanism of lipid binding of the Dvl2 DEP domain. Due to the presence of a large basic patch on its surface, it is possible that the DEP domain recognizes a pair (or more) of lipids at the membrane. A combination of NMR titrations, liposome-binding assays, tryptophan fluorescence, and surface plasmon resonance analyses in a range of physiological pH values will be used to define the Dvl2 DEP domain lipid-binding properties. Since the Dvl PDZ weakly binds to the Fz receptor, the contribution of the DEP domain in this interaction will also be investigated. With the purpose of using membrane mimetics, he has found that the Dvl2 DEP domain is stable in sodium cholate micelles from NMR analysis. Consequently, in Specific Aim 2, the spectrum of affinities of the DEP domain for PA (and other lipids ligands identified in Specific Aim 1) embedded in micelles will be measured to develop a quantitative basis for predicting subcellular targeting. The cooperative effects of headgroup ligation and hydrophobic insertion will be defined, as will liposome-binding properties. Membrane penetration of DEP will be elucidated with paramagnetic spin labels and by tryptophan fluorescence analysis with quenchers. Determinants of membrane association will be kinetically characterized using surface plasmon resonance. Understanding the structural basis of phospholipid recognition by the Dvl2 DEP domain would enable us to derive mechanistic insights, design mechanism-based inhibitors, create functionally-specific mutations, and precisely manipulate the Wnt/PCP pathway. NARRATIVE: The Wnt signaling pathway describes the activation of several distinct networks of proteins characterized for their roles in embryogenesis such as axis formation, nervous system patterning, and coordination of cell behavior, as well as for their implications in cancer development. Equilibrium among the different branches of the Wnt signaling pathway depends upon the subcellular localization of its participant proteins. By molecularly defining protein interactions in the Wnt pathway, the investigator should be able to rationally manipulate subcellular membrane targeting of host proteins.